Methods and Devices for Detection and Measurement of Analytes

ABSTRACT

Sensors for target entities having functionalized thereon, at least one aptamer specific to the target entity, and methods of making and using the same are described for use in glycated protein monitoring and/or biomarkers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.14/375,929, filed under 35 U.S.C. §371 on Jul. 31, 2014, now allowed;which is a national stage application of international applicationPCT/US13/24158, filed under the authority of the Patent CooperationTreaty on Jan. 31, 2013, published; which claims the benefit of U.S.Provisional Application No. 61/593,054, filed under 35 U.S.C. §111(b) onJan. 31, 2012. The entire disclosures of all the aforementioned priorityapplications are expressly incorporated herein by reference for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was not made with any government support and thegovernment has no rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jan. 31, 2013, is named420_53354_SEQ_LIST_D2012-15.txt, and is 3,888 bytes in size.

TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY

The present invention relates to aptamer functionalized surface plasmonresonance (SPR) sensors, methods of making and methods of using thesame.

BACKGROUND

The following description provides a summary of information relevant tothe present disclosure and is not a concession that any of theinformation provided or publications referenced herein is prior art tothe claimed invention.

The direct detection of blood proteins can benefit a number ofscientific and clinical applications, such as in monitoring the ratio ofspecific protein glycation in diabetes, biomarkers for drug research andenvironmental monitoring, cancer diagnostics and treatment, and thelike. The current clinical and laboratory measurement techniques forblood proteins are boronate affinity immunoassay, high-performanceliquid chromatography (HPLC), mass spectrometry and capillary basedsystems, which are time consuming and costly.

More efficient and fast response measurement methods could greatlybenefit and enhance related application areas, especially for developingthe next generation of portable handheld diagnostic devices capable ofreal-time analysis. Several optics-based diagnostic techniques, such asnear-infrared spectroscopy, polarimetry, optical coherence tomography,surface plasmon resonance (SPR), Raman and fluorescence spectroscopyhave recently been investigated for monitoring blood components. Many ofthese optical methods, however, are limited in their usefulness due tothe effects of confounding substances that may be present in the sampleunder investigation.

One method for the in vitro selection of nucleic acid molecules that areable to bind with high specificity to target molecules is generallyknown as SELEX (Systematic Evolution of Ligands by ExponentialAmplification) and is described in U.S. Pat. No. 5,475,096 titled“Nucleic Acid Ligands” and U.S. Pat. No. 5,270,163, titled “Nucleic AcidLigands” each of which is specifically incorporated by reference herein.

Although the currently used SELEX processes are useful, there is alwaysa need for improved processes that allow for the selection of moreselective of aptamers to be generated from in vitro selectiontechniques.

SUMMARY

In one broad aspect, there is provided herein a sensor for detecting thepresence of a target entity, comprising an aptamer probe having anamine-terminated end or similar linked to a substrate, wherein, when thesensor is excited by an energy source either: i) in the absence ofspecific interaction between the target entity and the aptamer probe, abaseline signal is emitted; or ii) in the presence of specificinteraction between the target entity and the aptamer probe, a detectionsignal is emitted, wherein the baseline signal is different from thedetection signal, whereby the selective presence of the target entity isdetected.

In certain embodiments, the aptamer probe includes a nucleotide sequencewhich specifically interacts with the target entity.

In certain embodiments, the target entity is one or more of: a largebiomolecule, a small biomolecule, an organic molecule, a small molecule,a nucleic acid, a metal ion, a protein, an enzyme, a peptide, a drug, adye, a cancer cell, a virus, a hormone, or a microorganism. In certainembodiments, the protein is a blood protein.

In certain embodiments, the aptamer probe comprises an aptamer and anattached amine moiety.

In certain embodiments, the aptamer probe includes a SAM linker betweenthe substrate and the amine moiety.

In certain embodiments, the amine-terminated aptamer probe is linked tothe substrate by 3-mercaptopropionic acid (MPA).

In certain embodiments, the sensor has a tunable detectable rangecapable of pM to nM detection, based on the linker characteristics.

In broad aspect, there is provided herein a method of determining if atarget entity is present in a sample comprising: i) contacting thesample with a sensor as described herein; ii) exciting the sensor withan energy source; and, iii) determining the strength of emitted signal,thereby determining whether the target entity is present in the sample.

In certain embodiments, the energy source is measured using surfaceplasmon resonance (SPR).

In certain embodiments, the method has a response time of less than 1minute. In certain embodiments, the method has a response time of lessthan 1 minute at about ambient temperature.

In another broad aspect, there is provided herein a kit for thedetection of a target entity, comprising: a sensor as described herein;and at least one container containing the sensor, where a sample may beadded to the container.

In another broad aspect, there is provided herein a method for making asensor, comprising: i) immobilizing a self-assembled monolayer (SAM)linker to a substrate; and ii) immobilizing an amine-terminated aptamerto the SAM linker.

In another broad aspect, there is provided herein a method for making asensor of, comprising: i) functionalizing a substrate with aself-assembled monolayer (SAM) linker; ii) exposing the functionalizedsubstrate of step i) to a composition having an amine moiety sufficientfor the amine moiety to be immobilized on the SAM linker; iii) exposingthe amine-functionalized substrate of step ii) to at least one aptamersufficient for the aptamer to be immobilized on the amine moiety; iv)optionally, removing non-specifically immobilized aptamer; and v)exposing the amine-terminated aptamer functionalized substrate of stepiii) or iv) to a blocking agent sufficient to block non-occupied SAMsites activated by the amine moiety.

In certain embodiments, the composition having the amine moiety iscoupled to one or more of: N-hydroxysuccinimide (NHS) andN-(3-dimethylamnopropyl)-N-ethylcarbodiimide hydrochloride (EDC).

In another aspect, there is provided herein a method for detecting bloodproteins using a sensor as described herein.

In another aspect, there is provided herein a method for theultrasensitive and selective detection and measurement of glycatedproteins for application in diabetes therapeutic guidance. In oneparticular embodiment, the method includes the use of surface plasmonresonance spectroscopy.

In another embodiment, this functionalization method is applicable toother sensing modalities including Raman and fluorescence spectroscopy,and can be used to further improve performance of existing monitoringtechnologies.

In another aspect, there is provided herein a method to optimize the invitro selection of aptamers to target specific glycated forms of bloodproteins. In one embodiment, a surface functionalization method is usedto optimize the sensitivity and selectivity based on the targetcharacteristics.

In another aspect, there is provided herein a method to further reduceeffects of confounding substances that may be present in the sampleunder investigation.

In yet another aspect, there is provided herein a robust, low cost, andportable sensing platform that is capable of achieving similarperformance to existing large scale clinical instrumentation. Inaddition, the integrated platform is useful in a diagnostic devicecapable of assessing compliance to insulin dependent diabetes therapy.The integrated platform allows for a low cost handheld device that canbe used in either a physician's office or in a home environment. Theintegrated platform also provides an immediate analysis of the datagathered, thus allowing the caregiver and/or patient to assess thepatient's long-term glucose regulation compliance.

In another broad aspect, there is provided herein a method foridentifying aptamers targeted to a defined site (e.g., glycated proteinsite), comprising introducing a non-target candidate (e.g., non-glycatedcandidate) in an at least one round of a systematic evolution of ligandsby exponential (SELEX) enrichment protocol, and introducing thenon-target candidate in at least a second round of SELEX protocol toremove aptamer candidates with affinity to both glycated andnon-glycated protein forms.

In another broad aspect, there is provided herein a surfacefunctionalization method to optimize sensitivity and/or selectivitybased on target and/or aptamer characteristics, comprising: using abinary self-assembled monolayer (SAM) formation process using linkageshaving a desired linking spacing and/or length, wherein at least one ofthe linkage spacing and/or length are chosen in order to optimizesurface plasmon resonance (SPR) sensitivity and selectivity based ontarget and/or aptamer characteristics.

In another aspect, there is provided herein a method for optimizingsensitivity and/or selectivity of a sensor for one or more analytes,comprising linking one or more types of aptamers to a substrate with aself-assembled monolayer (SAM) linkage, the SAM linkage having a desiredlinking spacing and/or length to form a functionalized surface on thesubstrate. The desired linkage spacing and/or length can be chosen inorder to optimize at least one of surface plasmon resonance (SPR), Ramanspectroscopy, or fluorescence spectroscopy sensitivity and selectivitybased on analyte and/or aptamer characteristics.

In certain embodiments, at least one packing density and/or length ofthe SAM linkage affects a surface plasmon resonance (SPR) signal.

In certain embodiments, linkage is through a binary SAM and reductivedesorption process.

In certain embodiments, the desorption process comprising exposing thefunctionalized surface of the substrate to a material resistant toprotein adsorption to prevent non-specific adsorption of protein on thefunctionalized substrate.

In certain embodiments, the protein adsorption resistant materialcomprises 1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3).

In certain embodiments, the SAM linkage comprises using a thiol SAMimmobilization method, wherein a thiol compound has a carboxy moietycapable of forming a stable bond with the aptamer.

In certain embodiments, the thiol compound comprisesdithiobis-N-succinimidyl propionate (DTSP).

In certain embodiments, the SAM linkage is formed usingdithiobis-N-succinimidyl propionate (DTSP) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3), wherein PEG3prevents non-specific adsorption of proteins, and wherein a carboxylicmoiety on DTSP forms a stable bonding with the aptamer.

In certain embodiments, a binary SAM thiol solution is used in the SAMlinkage.

In certain embodiments, the binary SAM thiol solution is prepared bymixing 1 mM ethanol solutions of 3-mercaptopropionic acid (MPA) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3), while keeping atotal concentration of the binary SAMs at about 1 mM.

In certain embodiments, MPA and PEG3 are present at ratio of: about20:80, about 50:50 or about 80:20.

In certain embodiments, the method further comprises eliminating MPA byreductive desorption, leaving PEG3 intact; and allowingdithiobis-N-succinimidyl propionate (DTSP) to a covalent bond with anamino group on the aptamer, wherein the aptamer attaches to DTSP only,and wherein while PEG3 does not form any bond.

In certain embodiments, the aptamer comprises an amine-modified aptamercapable of being immobilized onto the MPA.

In certain embodiments, the surface has an optimal dynamic in the rangeof about 5 nM to about 1000 nM.

In certain embodiments, the sensor includes a mixed length spacer layer.

In certain embodiments, the mixed length layer comprises11-mercaptoundecanoic acid (MUA) combined with 3-mercaptopropionic acid(MPA).

In certain embodiments, a water soluble thiol-containing amino acidcapable of directly binding to the surface to form the self-assemblymonolayer (SAM) is used. In certain embodiments, the amino acidcomprises cysteine.

In another aspect, there is provided herein a method for forming asensor for one or more analytes, comprising: adsorbing binary componentscomprised of 3-mercaptopropionic acid (MPA) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3) on a substrate;reductively desorbing MPA from the substrate of step a); immersing thesubstrate of step b) in a DTSP solution to form a DTSP layer on thesubstrate; immobilizing at least one type of aptamer on the substrate ofstep c); and, removing unbound aptamer from the PEG3 on the substrate ofstep d), thus leaving aptamer attached to the DTSP layer of thesubstrate.

In another aspect, there is provided herein a method for forming asensor for one or more analytes, comprising: adsorbing binary componentscomprised of 3-mercaptopropionic acid (MPA) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3) on a gold surfacesubstrate in an ethanol solution; reductively desorbing MPA from thesubstrate in a 0.5 M KOH solution, wherein the adsorbed MPA in aphase-separated binary self-assembled monolayer (SAM) of MPA and PEG3 isselectively reduced by applying a potential of −1.2 V for 30 min to thesolution; immersing the substrate having the PEG3 layer thereon, in a 1mM DTSP solution to form a DTSP layer thereon; immobilizing at least onetype of aptamer on the substrate; and removing aptamer from the PEG3 onthe substrate, thus leaving aptamer attached to the DTSP layer of thesubstrate.

In certain embodiments, the substrate has a gold surface.

In certain embodiments, the analyte comprises a glycated form of aprotein in blood or serum.

In certain embodiments, the method comprises determining a fraction of aspecific glycated protein from a total serum protein level.

In certain embodiments, the analyte comprises one or more non-glycatedand/or glycated forms of human hemoglobin, albumin, including humanserum albumin (HSA), immunoglobulin G (IgG), immunoglobulin M (IgM),fibrinogen, and/or fragments thereof.

In certain embodiments, the analytes comprise at least a first analytehaving a different half-life from at least a second analyte, and themethod further comprises quantifying the first and second analytes toprovide a retrospective judgment regarding levels of the first andsecond analytes over one or more time periods.

In certain embodiments, the first analyte comprises hemoglobin and thesecond analyte comprises IgM.

In certain embodiments, the analytes comprise at least a first analyte,at least a second analyte and at least a third analyte, each of thefirst, second and third analytes having different half-lives, the methodfurther comprising: quantifying the first, second and third analytes toprovide a retrospective judgment regarding levels of the first, secondand third analytes over one or more time periods.

In certain embodiments, the first analyte comprises hemoglobin, thesecond analyte comprises IgM and the third analyte comprises albumin.

In certain embodiments, the method is useful for monitoring past averageglucose levels, the method comprising: contacting a sensor formed by amethod described herein with a blood sample; determining an amount ofthe glycated form of the protein in the blood; and correlating an amountof the protein present in the blood sample in the glycated form to acontrol glucose level for a given time frame.

In certain embodiments, the amount of the glycated form of the proteinis determined using surface plasmon resonance (SPR).

In another aspect, there is provided herein a sensor for detecting thepresence of one or more analytes, wherein the sensor is formed by anyone of the methods described herein.

In certain embodiments, the aptamer includes a nucleotide sequencecapable of interacting with a specific analyte.

In certain embodiments, the sensor is capable of interacting with one ormore analytes selected from: a large biomolecule, a small biomolecule,an organic molecule, a small molecule, a nucleic acid, a metal ion, aprotein, an enzyme, a peptide, a drug, a dye, a cancer cell, a virus, ahormone, or a microorganism.

In certain embodiments, the analyte is one or more of: a biologicalsample, an environmental sample, a chemical sample, a pharmaceuticalsample, a food sample, an agricultural sample, and a veterinary sample.

In certain embodiments, the protein is a blood protein.

In certain embodiments, the sensor has a tunable detectable rangecapable of pM to nM detection, based on the linker characteristics.

In certain embodiments, the sensor has a response time of less than 1minute.

In certain embodiments, the sensor has a response time of less than 1minute at about room temperature.

In another aspect, there is provided herein a kit for the detection ofone or more analytes, comprising: a sensor as described herein; and atleast one container containing the sensor, where a sample may be addedto the container.

In another aspect, there is provided herein a method for reducing aneffect of at least one confounding substance that may be present in asample, comprising: incorporating one or more hydrophilic groups innon-binding locations on the substrate sufficient to substantiallyreduce/prevent non-specific adsorption of the confounding substance,linking an aptamer to the substrate with a self-assembled monolayer(SAM) linkage, the SAM linkage having a desired linking spacing and/orlength to form a functionalized surface on the substrate, and detectingaptamer binding response by SPR sensor at separation distance beyondnormal SPR detection limit.

Also described herein is a method which uses surface plasmon resonance(SPR) spectroscopy and custom developed aptamer-based functionalizedsensor surfaces to detect and/or quantify one or more target molecules,or fragments thereof, in a test environment. The method allows for thedetection and/or measurement of such molecules with a large range ofhalf-lives, including but not limited to target molecules withhalf-lives shorter than that of hemoglobin. Furthermore, the method canbe conducted without the use of tags or labels such as fluorescent dyes,or photocrosslinking. The method also has low sample consumption, andprovides a fast response time (generally seconds), making it useful forapplication in assessing glycemic compliance.

In another aspect, there is provided herein a sensor, comprising: one ormore types of aptamers linked to a substrate with a self-assembledmonolayer (SAM) linkage, the SAM linkage having a desired linkingspacing and/or length to form a functionalized surface on the substrate,the desired linkage spacing and/or length being chosen in order tooptimize at least one of surface plasmon resonance (SPR), Ramanspectroscopy, or fluorescence spectroscopy sensitivity and selectivitybased on analyte and/or aptamer characteristics.

In certain embodiments, the at least one packing density and/or lengthof the SAM linkage affects a surface plasmon resonance (SPR) signal.

In certain embodiments, the linkage is through a binary SAM andreductive desorption process.

In certain embodiments, the functionalized surface of the substrate hasbeen exposed to a material resistant to protein adsorption sufficient toinhibit non-specific adsorption of protein on the functionalizedsurface.

In certain embodiments, the protein adsorption resistant materialcomprises 1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3).

the SAM linkage comprises a thiol compound having a carboxy moietycapable of forming a stable bond with the aptamer.

In certain embodiments, the thiol compound comprisesdithiobis-N-succinimidyl propionate (DTSP).

In certain embodiments, the SAM linkage is formed usingdithiobis-N-succinimidyl propionate (DTSP) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3), wherein PEG3prevents non-specific adsorption of proteins, and wherein a carboxylicmoiety on DTSP forms a stable bonding with the aptamer.

In certain embodiments, a binary SAM thiol solution is used in the SAMlinkage.

In certain embodiments, the binary SAM thiol solution is prepared bymixing 1 mM ethanol solutions of 3-mercaptopropionic acid (MPA) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3), while keeping atotal concentration of the binary SAMs at about 1 mM.

In certain embodiments, the MPA and PEG3 are present at ratio of: about20:80, about 50:50 or about 80:20.

In certain embodiments, the MPA has been eliminated by reductivedesorption, leaving PEG3 intact; and dithiobis-N-succinimidyl propionate(DTSP) has bonded with an amino group on the aptamer, and PEG3 does notform any bond.

In certain embodiments, the aptamer comprises an amine-modified aptamercapable of being immobilized onto of 3-mercaptopropionic acid (MPA).

In certain embodiments, the surface has an optimal dynamic in the rangeof about 5 nM to about 1000 nM.

In certain embodiments, the sensor includes a mixed length spacer layer.

In certain embodiments, the mixed length layer comprises11-mercaptoundecanoic acid (MUA) combined with 3-mercaptopropionic acid(MPA).

In certain embodiments, the SAM linkage comprises a water solublethiol-containing amino acid capable of directly binding to the surfaceof the substrate.

In certain embodiments, the amino acid comprises cysteine.

In another aspect, there is provided herein a sensor where at least thesurface of the substrate is gold.

In certain embodiments, the sensor is configured for sensing an analytecomprised of a glycated form of a protein in blood.

In certain embodiments, the sensor is configured for determining afraction of a specific glycated protein from a total serum proteinlevel.

In certain embodiments, the analyte comprises one or more of: humanhemoglobin, albumin, including human serum albumin (HSA), immunoglobulinG (IgG), immunoglobulin M (IgM), fibrinogen, and/or fragments thereof,the analyte being in glycated or non-glycated forms.

In certain embodiments, the analytes comprise at least a first analytehaving a different half-life from at least a second analyte.

In certain embodiments, the first analyte is comprised of hemoglobin andthe second analyte is comprised of immunoglobulin M (IgM); and, whereineither of the first analyte or the second analyte is present in aglycated form or a non-glycated form.

In certain embodiments, the analytes comprise at least a first analyte,at least a second analyte and at least a third analyte, each of thefirst, second and third analytes having different half-lives.

In certain embodiments, the first analyte is comprised of hemoglobin,the second analyte is comprised of IgM, and the third analyte iscomprised of albumin; wherein one or more of the first analyte, thesecond analyte or the third analyte is present in a glycated form or anon-glycated form.

In another aspect, there is provided herein a use of any one of thesensors described herein for monitoring past average blood analytelevels, by: contacting a sensor formed by a method described herein witha blood sample; determining an amount of the glycated form of theanalyte in the blood; and correlating an amount of the analyte presentin the blood sample analyte in a glycated form of to a control level fora given time frame.

In certain embodiments, the amount of the glycated form of the proteinis determined using surface plasmon resonance (SPR).

In certain embodiments, the aptamer includes a nucleotide sequencecapable of interacting with a specific analyte.

In certain embodiments, the sensor is capable of interacting with one ormore analytes selected from: a large biomolecule, a small biomolecule,an organic molecule, a small molecule, a nucleic acid, a metal ion, aprotein, an enzyme, a peptide, a drug, a dye, a cancer cell, a virus, ahormone, or a microorganism.

In certain embodiments, the analyte is one or more of: a biologicalsample, an environmental sample, a chemical sample, a pharmaceuticalsample, a food sample, an agricultural sample, and a veterinary sample.

In certain embodiments, the analyte is a blood protein.

In certain embodiments, the sensor has a tunable detectable rangecapable of pM to nM detection, based on the linker characteristics.

In certain embodiments, the sensor has a response time of less than 1minute.

In certain embodiments, the sensor has a response time of less than 1minute at about ambient temperature.

In certain embodiments, the sensor includes an aptamer where the aptamercomprises a DNA sequence having at least 70% identity to the entiresequence of any one of SEQ ID NOS: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14and 15.

In another aspect, there is provided herein a kit for the detection ofone or more analytes, comprising: any one or more of the sensorsdescribed herein; and at least one container including the sensor, wherea sample may be added to the container.

In certain embodiments, the kit further comprises one or more solidsupports, one or more separating agents for separating the sensor froman elute, and one or more reagents for separating an aptamer from thesensor.

In another aspect, there is provided herein a method of identifying asingle target-site binding aptamer from a pool of nucleic acids havingsingle-target-site-binding-aptamers andnon-target-protein-binding-aptamers therein, comprising:

-   -   a) adding to the pool of nucleic acids, a        single-site-target-protein-complex, wherein both the        single-target-site-binding-aptamers and the        non-target-protein-binding-aptamers present in the pool bind to        the single-site-target-protein-complex, and form a        single-target-site-binding-aptamer+non-target-protein-binding-aptamer+single-site-target-protein-complex;    -   b) separating the        single-target-site-binding-aptamer+non-target-protein-binding-aptamer+single-site-target-protein-complex        from the pool;    -   c) eluting the single-target-site-binding-aptamers and the        non-target-protein-binding-aptamers from the        single-site-target-protein-complex;    -   d) adding to the elute of the previous step, a        non-target-protein-complex, wherein the        non-target-protein-binding-aptamers present in the elute of        step c) bind to the non-target-protein-complex, and form a        non-target-protein-binding-aptamer+non-target-protein-complex;    -   e) separating the        non-target-protein-binding-aptamer+non-target-protein-complex        from the elute of the previous step, leaving the        single-target-site-binding-aptamer in the elute; and,    -   f) separating the single-target-site-binding-aptamers from the        elution; optionally, further amplifying the        single-target-site-binding-aptamers.

In certain embodiments, the single-target-site-binding-aptamers are usedto select for one of: hemoglobin, immunoglobulin G (IgG), immunoglobulinM (IgM) and albumin.

In certain embodiments, the single-target-site-binding-aptamers areselected from: SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and15.

In certain embodiments, the single-site-target-protein is immobilized ona solid support.

In certain embodiments, the non-target-protein complex is immobilized ona solid support.

In certain embodiments, the solid support comprises a magnetic bead, achromatographic matrix, a microtiter dish or an array.

In another aspect, there is provided herein an aptamer that binds toglycated hemoglobin, wherein the aptamer comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 4 and 5.

In certain embodiments, the glycated hemoglobin is human hemoglobin.

In certain embodiments, the aptamer has a dissociation constant forhuman hemoglobin of 100 nM or less.

In another aspect, there is provided herein an aptamer described hereinthat binds to glycated hemoglobin, wherein the aptamer comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:4 and 5, and one or more of: a 5′-linker and a 3′-linker.

In certain embodiments, the linker is a self-assembled monolayer (SAM).

In another aspect, there is provided herein, an aptamer with at least70% identity to the entire sequence of any one of SEQ ID NOs: 4 and 5and that binds to human glycated hemoglobin.

In another aspect, there is provided herein a composition of mattercomprising a self-assembled monolayer (SAM) conjugated to a nucleic acidaptamer molecule comprising a polynucleotide sequence capable ofspecifically binding a region of glycated hemoglobin, wherein thepolynucleotide sequence is selected from the group consisting of SEQ IDNOs:4 and 5.

In another aspect, there is provided herein an aptamer that binds tonon-glycated hemoglobin, wherein the aptamer comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 6 and 7.

In certain embodiments, the non-glycated hemoglobin is human hemoglobin.

In certain embodiments, the aptamer has a dissociation constant forhuman hemoglobin of 100 nM or less.

In another aspect, there is provided herein aptamer that binds tonon-glycated hemoglobin, wherein the aptamer comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:6 and 7, andone or more of: a 5′-linker and a 3′-linker.

In certain embodiments, the linker is a self-assembled monolayer (SAM).

In another aspect, there is provided herein an aptamer with at least 70%identity to the entire sequence of any one of SEQ ID NOs:6 and 7 andthat binds to human non-glycated hemoglobin.

In another aspect, there is provided herein a composition of mattercomprising a self-assembled monolayer (SAM) conjugated to a nucleic acidaptamer molecule comprising a polynucleotide sequence capable ofspecifically binding a region of non-glycated hemoglobin, wherein thepolynucleotide sequence is selected from the group consisting of SEQ IDNOs:6 and 7.

In another aspect, there in provided herein an aptamer that binds toglycated serum albumin, wherein the aptamer comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:3, 8 and 9.

In certain embodiments, the glycated serum albumin is human glycatedserum albumin.

In certain embodiments, the aptamer has a dissociation constant forhuman glycated serum albumin of 100 nM or less.

In another aspect, there is provided herein an aptamer that binds toglycated serum albumin, wherein the aptamer comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:3, 8 and 9,and one or more of: a 5′-linker and a 3′-linker.

In certain embodiments, the linker is a self-assembled monolayer (SAM).

In another aspect, there is provided herein an aptamer with at least 70%identity to the entire sequence of any one of SEQ ID NOS:3, 8 and 9, andthat binds to human glycated serum albumin.

In another aspect, there is provided herein a composition of mattercomprising a self-assembled monolayer (SAM) conjugated to a nucleic acidaptamer molecule comprising a polynucleotide sequence capable ofspecifically binding a region of glycated serum albumin, wherein thepolynucleotide sequence is selected from the group consisting of SEQ IDNOs:3, 8 and 9.

In another aspect, there is provided herein an aptamer that binds tonon-glycated serum albumin, wherein the aptamer comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:10, 11, 12,13, 14 and 15.

In certain embodiments, the non-glycated serum albumin is human glycatedserum albumin.

In certain embodiments, the aptamer has a dissociation constant forhuman non-glycated serum albumin of 100 nM or less.

In another aspect, there is provided herein an aptamer that binds tonon-glycated serum albumin, wherein the aptamer comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 10, 11, 12,13, 14 and 15, and one or more of: a 5′-linker and a 3′-linker.

In certain embodiments, the linker is a self-assembled monolayer (SAM).

In another aspect, there is provided herein an aptamer with at least 70%identity to the entire sequence of any one of SEQ ID NOS: 10, 11, 12,13, 14 and 15, and that binds to human non-glycated serum albumin.

In another aspect, there is provided herein a composition of mattercomprising a self-assembled monolayer (SAM) conjugated to a nucleic acidaptamer molecule comprising a polynucleotide sequence capable ofspecifically binding a region of non-glycated serum albumin, wherein thepolynucleotide sequence is selected from the group consisting of SEQ IDNos. 10, 11, 12, 13, 14 and 15.

In certain embodiments, the aptamer comprises at least one chemicalmodification.

In certain embodiments, the modification is selected from the groupconsisting of: a chemical substitution at a sugar position, a chemicalsubstitution at an internucleotide linkage, and a chemical substitutionat a base position.

In another aspect, there is provided herein a test reagent comprising aneffective amount of an aptamer described herein, or a salt thereof, anda support therefor.

In another aspect, there is provided herein a kit comprising at leastone aptamer as described herein.

In certain embodiments, the aptamer is PEGylated.

In certain embodiments, the PEGylated aptamer molecule includes1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3).

In certain embodiments, the SAM linkage is formed usingdithiobis-N-succinimidyl propionate (DTSP) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3).

In certain embodiments, the aptamer comprises at least one chemicalmodification.

In certain embodiments, the modification is selected from the groupconsisting of: a chemical substitution In certain embodiments, at asugar position, a chemical substitution at an internucleotide linkage,and a chemical substitution at a base position.

A test reagent comprising an effective amount of one or more aptamersdescribed herein or a salt thereof, and a support therefor.

A kit comprising one or more aptamers described herein.

In another aspect, there are provided herein purified and isolatednon-naturally occurring DNA sequences selected from the group consistingof SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

In another aspect, there is provided herein a method for reducing aneffect of at least one confounding substance that may be present in asample, comprising: a) incorporating one or more hydrophilic groups innon-binding locations on the substrate sufficient to substantiallyreduce/prevent non-specific adsorption of the confounding substance, b)linking an aptamer to the substrate with a self-assembled monolayer(SAM) linkage, the SAM linkage having a desired linking spacing and/orlength to form a functionalized surface on the substrate, and c)detecting aptamer binding response by SPR sensor at separation distancebeyond normal SPR detection limit.

Other systems, methods, features, and advantages of the presentinvention will be or will become apparent to one with skill in the artupon examination of the following drawings and detailed description. Itis intended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe present invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIG. 1: Schematic diagram of a sensing surface functionalization method.

FIG. 2: Nyquist plots of impedance spectra obtained in 100 mM PBsolution (pH 7.2) containing 5 mM Fe(CN)₆ ³⁻/Fe(CN)₆ ⁴⁻ (Column A) BareAu; (Column B) Au/MPA/EDC-NHS/EA/PPA; (Column C)Au/MPA/EDC-NHS/EA/PPA/APT1. The right plot shows the ₍R_(et)) of eachlayer. Impedance spectra were collected in the frequency range from 0.1Hz to 100 kHz with a potential amplitude of 5 mV rms at 10 points perdecade.

FIG. 3: Graph showing aptamer/thrombin binding ratio in mol by themagnetic beads coupling method.

FIG. 4: Graphs showing SPR response of bare Au and aptamer-modifiedsensors. All data points were averaged from 3 experimental datareadings. Samples were thrombin only (top plot) and thrombin with 400 nMBSA (bottom plot). The inlay plots are same data plotted on logarithmicscale to allow for better visualization at lower concentrations.

FIG. 5: Graph showing SPR responses of different sensing surfaces for400 nM BSA (BSA group), 500 nM thrombin (Thrombin group), and 500 nMthrombin with 400 nM BSA (Thrombin+BSA group). The error bars representthe standard deviation of the values determined from three freshlyprepared samples.

FIG. 6: Graph showing SPR responses of different sensing surfaces for 50nM, 250 nM, 500 nM thrombin with and without 400 nM BSA, upper axis(APT1), lower axis (APT2); the zero position of lower axis has beenshifted intentionally to better distinguish between data points thatwould be overlapping.

FIG. 7: Schematic illustration of an excited surface plasmon.

FIG. 8a : Schematic illustration of SPR with Kretschmann configuration.

FIG. 8b : Schematic illustration of shift in resonance angle due tochange in refractive index.

FIG. 9: Schematic illustration of binding HbA1c with aptamer immobilizedon a SAM surface attached to a SPR sensing surface (top); and, schematicillustration of shift in resonance angle due to change in refractiveindex (bottom).

FIG. 10: Graphs showing SPR response for HSA at different glycationlevels (% percent ratios; glycated/total protein). Note: the totalprotein concentration of each sample is constant at a level of 1 μg/mLtotal protein. (Green) Aptamer functionalized surface (Red) Bare-Ausurface.

FIGS. 11a-11e : Schematic illustration of reductive desorption forDTSP-PEG3 binary SAM formation: (FIG. 11a ) co-adsorption of MPA andPEG3 on Au; (FIG. 11b ) reductive desorption of MPA; (FIG. 11c )adsorption of DTSP; (FIG. 11d ) aptamer immobilization; and, (FIG. 11e )removing aptamer from PEG3.

FIG. 12: Schematic illustration of a MB counter-selection for a SELEXprocess.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

DEFINITIONS

All publications, published patent documents, and patent applicationscited in this specification are indicative of the level of skill in theart(s) to which the invention pertains. All publications, publishedpatent documents, and patent applications cited herein are herebyincorporated by reference to the same extent as though each individualpublication, published patent document, or patent application wasspecifically and individually indicated as being incorporated byreference.

As used in this specification, including the claims, the singular forms“a,” “an,” and “the” include plural references, unless the contentclearly dictates otherwise, and are used interchangeably with “at leastone” and “one or more.” That is, a reference to “an aptamer” includesmixtures of aptamers, reference to “nucleic acids” includes mixtures ofnucleic acids, and the like.

As used herein, the term “about” represents an insignificantmodification or variation of the numerical values such that the basicfunction of the item to which the numerical value relates is unchanged.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that comprises,includes, or contains an element or list of elements does not includeonly those elements but may include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

The term “aptamers” as used here indicates oligonucleic acid or peptidemolecules that are capable to bind a specific target entity. In general,aptamers are artificial oligonucleotides which can serve as antibodymimics because of their high affinity and selectivity for various targetcompounds ranging from small molecules, such as drugs and dyes, tocomplex biological molecules such as enzymes, peptides, and proteins.Custom aptamers can be identified from random oligonucleotide librariesfor specific target compounds by an in vitro iterative process calledSystematic Evolution of Ligands by Exponential Amplification (SELEX).For examples of SELEX processes see U.S. Pat. Nos. 5,270,163; 5,475,096;and 5,567,588, which are incorporated herein by reference in theirentirety.

Aptamers can form a 3D structure serving as receptors specific to theirtarget compounds similar to antibodies. Aptamers also have a number ofadvantages over antibodies such as a tolerance to wide ranges of pH andsalt concentrations, heat stability, ease of synthesis, and costefficiency. The specificity and affinity of aptamers are comparable, ifnot higher, to antibodies. Aptamers are also capable of being reversiblydenatured for the release of target compounds, which makes the aptamersespecially useful receptors for biosensing applications.

For example, aptamers can be comprised of single-stranded (ss)oligonucleotides and/or be chemically synthesized peptides that havebeen engineered through repeated rounds of in vitro selection, orequivalent techniques identifiable by a skilled person, to bind tovarious targets.

An “aptamer” or “nucleic acid ligand” is a set of copies of one type orspecies of nucleic acid molecule that has a particular nucleotidesequence. An aptamer can include any suitable number of nucleotides.“Aptamers” refer to more than one such set of molecules. Differentaptamers may have either the same number or a different number ofnucleotides. Aptamers may be DNA or RNA and may be single stranded,double stranded, or contain double stranded regions.

It is to be understood that that affinity interactions between andaptamer and an analyte or target are a matter of degree. That is, the“specific binding affinity” of an aptamer for its target means that theaptamer binds to its target generally with a much higher degree ofaffinity than such aptamer may binds to other, non-target, components ina mixture or sample.

As used herein the term “amplification” or “amplifying” means anyprocess or combination of process steps that increases the amount ornumber of copies of a molecule or class of molecules.

As used herein, “pool” is a mixture of nucleic acids of differingsequence from which to select a desired ligand. The source of a pool canbe from naturally-occurring nucleic acids or fragments thereof,chemically synthesized nucleic acids, enzymatically synthesized nucleicacids or nucleic acids made by a combination of the foregoingtechniques. Modified nucleotides, such as nucleotides with a detectablelabel, reactive groups or other modifications, can be incorporated intothe pool. In certain embodiments, a SELEX process and/or the improvedSELEX method described herein can be used to produce a pool. A pool canalso comprise nucleic acids with one or more common structural moieties,such that the nucleic acids can be separated by structure, and not bychemical, size, or other separation method. As used herein, a pool isalso sometimes referred to as a “library” or a “candidate or nucleicacid mixture.” For example, an “RNA pool” refers to a candidate mixturecomprised of RNA.

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides of anylength, and such nucleotides may include deoxyribonucleotides,ribonucleotides, and/or analogs or chemically modifieddeoxyribonucleotides or ribonucleotides. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules.

The term “sensor” as used herein indicates a device that measures aphysical quantity and converts it into a signal which can be read by anobserver or by an instrument. As is understood, a sensor is calibratedagainst known standards. Accordingly, a sensor can be used to capture atarget entity by exploiting the affinity of aptamer to the targetentity, and can be detected using techniques identifiable by a skilledperson upon reading of the present disclosure.

The term “detect” or “detection” as used herein indicates thedetermination of the existence, presence or fact of a target or signalin a limited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate including aplatform and an array. Detection is “quantitative” when it refers,relates to, or involves the measurement of quantity or amount of thetarget or signal (also referred as quantitation), which includes but isnot limited to any analysis designed to determine the amounts orproportions of the target or signal. Detection is “qualitative” when itrefers, relates to, or involves identification of a quality or kind ofthe target or signal in terms of relative abundance to another target orsignal, which is not quantified. An “optical detection” indicatesdetection performed through visually detectable signals: spectra orimages from a target of interest or a probe attached to the target.

The term “labeling agent,” “label,” or “detectable moiety”, or“detectable element” or “detectable component” refers to one or morereagents that can be used to detect a target molecule/aptamer complex. Adetectable moiety or label is capable of being detected directly orindirectly.

The terms “target,” “target entity” and “analyte” may be used hereininterchangeably, and generally refer to a substance, compound orcomponent whose presence or absence in a sample has to be detected.Analytes include but are not limited to biomolecules and in particularbiomarkers. The term “biomolecule” as used herein indicates a substancecompound or component associated to a biological environment includingbut not limited to sugars, amino acids, peptides proteins,oligonucleotides, polynucleotides, polypeptides, organic molecules,haptens, epitopes, biological cells, parts of biological cells,vitamins, hormones and the like. The term “biomarker” indicates abiomolecule that is associated with a specific state of a biologicalenvironment including but not limited to a phase of cellular cycle,health and disease state. The presence, absence, reduction, upregulationof the biomarker is associated with and is indicative of a particularstate. The terms “polypeptides,” “peptides,” and “proteins” are intendedto encompass polymers of amino acids of any length, whether linear orbranched, that may or may not be modified naturally or by intervention,such as by glycosylation, lipidation, acetylation, phosphorylation,disulfide bond formation, conjugation, or other manipulation ormodification.

The term “solid support” means any substrate having a surface to whichmolecules may be attached, directly or indirectly, through eithercovalent or non-covalent bonds. The substrate materials may be naturallyoccurring, synthetic, or a modification of a naturally occurringmaterial. Solid support materials may include magnetic beads, or anyother materials that are capable of having one or more functionalgroups, such as any of an amino, carboxyl, thiol, or hydroxyl functionalgroup, for example, incorporated on its surface. The solid support maytake any of a variety of configurations ranging from simple to complexand can have any one of a number of shapes, including beads, disks,particles, plates, rods, strips, tubes, wells, and the like. The surfacemay be relatively planar (e.g., a slide), spherical (e.g., a bead),cylindrical (e.g., a column), or grooved.

The term “separating” means any process whereby one or more componentsof a mixture are separated from other components of the mixture. Forexample, aptamers bound to target molecules can be separated from othernucleic acids that are not bound to target molecules and from non-targetmolecules. That is, a separation process or step allows for theseparation of all the nucleic acids in a candidate mixture into at leasttwo pools based on their relative affinity and/or dissociation rate tothe target molecule. The separation process can be accomplished byvarious methods. For example, magnetic beads upon which target moleculesare conjugated can also be used to separate aptamers in a mixture. Asanother example, surface plasmon resonance (SPR) technology can be usedto separate nucleic acids in a mixture by immobilizing a target on asensor chip and flowing the mixture over the chip, wherein those nucleicacids having affinity for the target can be bound to the target, and theremaining nucleic acids can be washed away.

The term “sample” as used herein refers to a mixture, gas, or substancethat may or may not comprise a target or analyte. Samples include butare not limited to biological samples, such as blood, sputum, breath,urine, semen, saliva, amniotic fluid, meningeal fluid, glandular fluid,nipple aspirate, lymph fluid, bronchial aspirate, joint aspirate,synovial fluid, cellular extract, cerebrospinal fluid, homogenized solidmaterial from stool or tissue samples, bacterial culture, viral culture,or experimentally-separated fractions thereof.

The term “non-target” refers to molecules in a sample that form anon-specific complex with an aptamer. It will be appreciated that amolecule that is a non-target for a first aptamer may be a target for asecond aptamer. Similarly, a molecule that is a target for a firstaptamer may be a non-target for a second aptamer.

General Description

The methods and devices described provide a system that has both thedesired high sensitivity and specificity to be able to detect glycatedproteins in a desired test environment and at sensitive concentrations.

In a particular aspect, the method includes determining the fraction ofa specific glycated protein from the total serum protein level.Non-limiting examples of such proteins include: human hemoglobin,albumin (such as human serum albumin (HSA)), and IgM proteins.

Two common glycated proteins found in the body are hemoglobin A1c(HbA1c) and immunoglobulin M (IgM) (which is a basic antibody present onB cells). Both HbA1c and IgM have different half-lives in the body;e.g., ˜6-8 weeks for HbA1c, and ˜1 week for IgM. Therefore,quantification of these glycated proteins in serum provides aretrospective judgment regarding glycemic control over both a shorterand longer term. The present method overcomes one of the primaryshortcomings of other tests where only one type of glycated serumprotein could be detected; and consequently, any compliance assessmentwith regard to glucose control was limited to only one fixed timeperiod. It is also to be noted that the present method overcomes othershortcomings that limit the assay results, such as interferences fromhemoglobinopathies, hemolysis, and/or anemia.

It is to be understood that, in other certain embodiments of themethods/devices described herein, one or more other molecules, orfragments thereof, such as other glycated proteins, can be accuratelytested. Since the present method facilitates detection and measurementof glycated blood proteins other than hemoglobin or site-specific HbA1c,the method is also useful for other technologies for the evaluation ofglycemic control.

In certain embodiments, a targeted historic time record of glycatedproteins from a period of about a few days up to about six weeks can beachieved depending on the specific glycated protein evaluated becausedifferent glycated proteins have different half-lives in blood. Incontrast, prior tests are limited to assessing only one fixed timeperiod.

This method and the platform using such method are highly miniaturizedand are useful in a handheld device to provide real-time detection andanalysis.

The method has the requisite sensitivity to be useful in medical testingof analytes.

The method further allows for the assessment of different types ofproteins, such as glycated hemoglobin and other glycated forms of bloodproteins.

In one method described herein, surface plasmon resonance is used with ahighly functionalized aptamer sensing surface in order to provide anaccurate, rapid and a relatively inexpensive method to assess glycemiccompliance by measuring the levels of certain glycemic proteins in bloodserum.

Determination of Aptamers

The method described herein is useful to detect different types ofaptamers. In one embodiment, in order to isolate and identifyoligonucleotides (aptamers) specific to the hemoglobin, albumin, and IgMglycated/non-glycated proteins, a Systematic Evolution of Ligands byExponential (SELEX) enrichment protocol can be used.

While the standard SELEX protocol allows for the screening of ligandsthat are particular to a given protein of interest, described herein isan improved SELEX method which identifies secondary aptamers that arecapable of detecting and capturing both protein versions (i.e., glycatedand non-glycated forms), as further explained herein.

In one embodiment described herein, the identification of the secondaryaptamer is used to determine the percent glycation which can becorrelated to mean glucose levels for a given time frame.

Detection Platform: Protein Sensing and Surface Plasmon Resonance (SPR)Spectroscopy

For protein detection, self-assembled monolayers (SAMs) are used toattach specific aptamers to gold SPR sensing surfaces. SPR spectroscopyitself is related to a phenomenon that occurs at the interface betweenconductors and dielectrics. At this interface, surface plasmons canexist which are charge density oscillations in the electron structure.These surface plasmons are most commonly excited with light in thevisible to near-infrared spectrum. This excitation can occur either asfreely propagating surface plasmons in a continuous metal surface or asa localized effect through the use of metal based nanoparticlestructures. In one embodiment described herein, a freely propagatingsurface plasmon approach is used.

Briefly, valence electrons are disassociated from the atomic core, andin essence behave as an electron gas in the presence of an externalelectric field; it can be shown the surface plasmon is a bound wave witha corresponding wave vector equal to:

$\begin{matrix}{{k_{sp} = {\frac{2\; \pi}{\lambda}\sqrt{\frac{ɛ_{metal}ɛ_{sample}}{ɛ_{metal} + ɛ_{sample}}}}},} & (1)\end{matrix}$

where λ is the wavelength of light, and ∈_(metal) and ∈_(sample) are therelative permittivity constants of media, respectively. Therefore,energy transfer to the surface plasmon will occur (i.e., it will beexcited) if the incident light has an electric field vector with atransverse mode polarization component with an energy close to k_(sp).

As shown in FIG. 7, the incident light vector has a component, k_(x),which can be represented by the equation:

$\begin{matrix}{{k_{x} = {\frac{2\; \pi}{\lambda}n_{i}\sin \; \theta_{i}}},} & (2)\end{matrix}$

where n_(i) is the index of refraction of the incident medium and θ_(i)is the incident angle of the incoming light contacting the metalsurface. Surface plasmon resonance is highly sensitive to localvariations in the refractive index of the sample due to the dependenceof ∈_(metal) and ∈_(sample) to the wavelength λ of the incident light.Changes in the refractive index can be measured using a reflectancebased approach. The light reflected at the interface of two dielectricmedia, as shown in FIG. 7, generates an evanescent field with maximumintensity at the surface which will resonate with free electrons (i.e.surface plasmons). This results in light energy being transferred to thesurface plasmon with a corresponding reduction in the degree ofreflected light. The angle at which this decrease occurs is commonlycalled the resonance angle.

A Kretschmann instrumentation configuration used to measure theresonance angle is illustrated in FIG. 8a . In this configuration, lightpasses through a prism which is reflected at the glass-metal interface.An expanded version of the interaction at the metal-light interface isshown in FIG. 7. Any change in the refractive index at the metal/sampleinterface will result in a corresponding change or shift in theresonance angle, as illustrated in FIG. 8 b.

The present method overcomes the drawbacks of use of SPR by itself,which is often adversely affected by the issue of lack-of-specificity.In addition, in the use of SPR by itself, if the sensing analyte doesnot elicit at least a moderate change in refractive index, the SPR alsois also adversely affected by lack-of-sensitivity issues, as well.

The present method overcomes these adverse issues by using the selectiveaptamers described herein, and by using self-assembled monolayers (SAMs)with SPR. The present method provides such advantages as highsensitivity and selectivity, cost effectiveness, chemical and thermalstability, facile synthesis and storage.

The presently described aptamer based sensing method is especiallyuseful as a sensing element in biosensor applications. The nucleic acidnature of aptamers also renders the immobilization and regenerationeasier. In one embodiment of an SPR application, the receptors (i.e.,aptamers) are immobilized on solid substrates of various types forcapturing target analytes or molecules (see FIG. 9).

In addition, the presently described method and apparatus overcome pastproblems with nonspecific adsorption of proteins that had beenassociated with SAMs where such nonspecific adsorption was detrimentalto the sensor activity. In particular, the non-specific adsorption fromcomplex sample matrices, like blood, urine or other clinical samples,was a major factor that limited the sensitivity.

Other limiting factors were the biophysical and chemical properties ofthe adsorbed surface itself. In such SAMs, these properties needed to besuppressed so as to ensure specific affinity interaction with theanalyte of interest. Furthermore, proteins adsorbed on a SAM surface,partially lose their bioactivity due to conformational changes insecondary structure and/or non-optimal orientation and distribution onthe surface. Also the protocols for preparation of surfaces and theconditions of mass transport significantly influence the proteinadsorption response. Therefore, quantitative comparison of data obtainedfrom different laboratories was difficult, and often inaccurate.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. All publications, including patentsand non-patent literature, referred to in this specification areexpressly incorporated by reference. The following examples are intendedto illustrate certain preferred embodiments of the invention and shouldnot be interpreted to limit the scope of the invention as defined in theclaims, unless so specified.

Example 1 Materials

The identified aptamers were synthesized by Integrated DNA Technologies(Coralville, Iowa), including a 15 bp aptamer (APT1):5′-NH₂—(CH₂)₆-GGTTGGTGTGGTTGG-3′ [SEQ ID NO:1], and a 34 bp aptamer(APT2): 5′-NH₂—(CH₂)₆-CTATCAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′. [SEQ IDNO:2].

Tosylactivated magnetic beads (MBs) were purchased from Invitrogen(Carlsbad, Calif.). All other chemicals were purchased from SigmaAldrich (Carlsbad, Calif.) at the highest purity available. Aptamersolutions were prepared with 1M pH 8 phosphate buffer. The3-mercaptopropionic acid (MPA) solution was prepared in ethanol. Proteinsample solutions were prepared using a 0.1M pH 7.2 PBS buffer solutionwith 5 mM KCl and 1 mM MgCl₂. The phosphoric acid (PPA) used was 100 mM.All other solutions were prepared in deionized (DI) water.

Instrumentation

SPR measurements were performed using a commercial grade SensiQDiscovery system (ICx Technologies, Arlington, Va.) at 25° C. Thissensor is based on a Kretschmann configuration, in which the light froma light-emitting diode (LED) integrated with a prism is firstlypolarized and then internally reflected from a gold surface. The angleof light reflection and the relative intensity was measured with aphotodiode array. When the sample solution was applied to the sensingsurface, the SPR profile minimum (also known as the SPR angle) shiftedas a function of the refractive index of the loaded sample, giving areal time refractive index reading (although, by itself the sensor isnot specific/selective for any given target). The SPR response profilewas recorded by the SensiQ software and then processed within MATLAB®.

Electrochemical impedance spectroscopy (EIS) measurements were carriedout using a Gamry Reference 600 potentiostat (Warminster, Pa.) in 5 mMFe(CN)₆ ³⁻/Fe(CN)₆ ⁴⁻ solution with KCl as a supporting electrolyte. Allthe experiments were carried out at room temperature with the solutionspurged with nitrogen gas for 15 minutes and the nitrogen blanket wasmaintained during the experiments. The experiments were performed at 25°C. Impedance spectra were collected in the frequency range from 0.1 Hzto 100 kHz with a potential amplitude of 5 mVrms at 10 points perdecade. EIS results were analyzed by fitting the experimental impedancedata to electrical equivalent circuit models. Parameters of theelectrical-equivalent circuits were obtained by fitting the impedancefunction to the measured Bode and Nyquist plots with a complex nonlinearleast square (CNLS) program built into the Gamry EIS 300 electrochemicalimpedance spectroscope.

Aptamer binding capacity was determined as follows: 10 nmol of aminemodified aptamer was coupled to 10 mg washed magnetic beads (MB s) in ashaker incubator at 37° C. for 18 hours. The unoccupied binding siteswere blocked by Bovine Serum Albumin (BSA). The aptamers-coupled MBswere washed thoroughly, and then 10 nmol of thrombin was mixed with theaptamer-coupled MBs for 2 hours in a shaker at room temperature. Thecontrol group was prepared by exactly the same method except for theabsence of aptamers. The total and unbounded proteins were measured witha carboxyl functionalized SPR sensor provided by SensiQ.

To demonstrate the use an aptamer-based SPR sensor for detecting bloodproteins, thrombin and antithrombin aptamer were chosen. Gold slideswere prepared by physical vapor deposition (PVD) forming a 1 nm layer oftitanium and a 50 nm layer of gold onto pre-cleaned microscope coverslides. These were then washed by copious amounts of DI water andethanol. They were dried in nitrogen gas before usage.

To functionalize the gold slides, they were immersed in the 10 mM MPAsolution for 30 min and then washed with ethanol and DI water. After theslides were dried, then they were immersed in a solution ofN-hydroxysuccinimide (NHS) andN-(3-dimethylamnopropyl)-N-ethylcarbodiimide hydrochloride (EDC) (NHS0.2M, EDC 0.05M) for 30 min. The slides were then washed with DI waterand then immersed in the 5 μM aptamer solution. Finally, the slides wererinsed with the PBS buffer to flush off non-specifically adsorbedproteins. Then the slides were ready for measurement. In certainembodiments, this two-step surface functionalization process isapplicable not only in SPR, but also Raman and fluorescencespectroscopy. The surface functionalization process is schematicallyillustrated in FIG. 1.

Non-coated (i.e., no gold) SensiQ base sensors were custom modified withthe developed gold based SPR sensing surfaces. Specifically, freshlyprepared aptamer-immobilized gold substrates were coupled to thestripped sensors with index matching optical oil. This was followed bythen loading of 100 μL 1 M ethanolamine (EA) at a flow rate of 20 μL/minto block the non-occupied MPA sites activated by the EDC/NHS, followedby an injection of 100 μL of 100 mM phosphoric acid (PPA) at 50 μL/minto remove the non-specific binding. The running buffer was 0.1 M pH 7.2PBS. The sensor was first normalized with the buffer for 10 min, thenthe thrombin sample (25 μL) at concentrations of 5 nM, 25 nM, 50 nM, 250nM 500 nM, 1000 nM, 2000 nM were loaded at 5 μL/min Samples with BSAwere all prepared with 400 nM BSA. All data was recorded at 290 s, 300s, and 310 s after the sample injection and averaged. Sensorregeneration was performed by the injection of 100 μL PPA at 50 μL/minfollowed by washing with the running buffer.

Results for Example 1

EIS Measurement

The successful immobilization of each functionalized layer was confirmedthrough EIS measurements. FIG. 2 shows the Nyquist plots of impedancespectra at different electrodes. The bare gold electrode represented avery small circle at high frequencies, indicating a very low electrontransfer resistance to the redox probe dissolved in the electrolytesolution (curve A). When the MPA was immobilized on the electrode andtreated with EA and PPA, the electron transfer resistance (R_(et))increased to 125Ω, (curve B). Then, when 5 μM of the APT1 aptamer wasadded and bound with the SAM, R_(et) increased to 600Ω (curve C). Inthis embodiment, the reactive sites on the gold electrode were blockedby EA (ethanolamine) to prevent non-specific adsorption of aptamers ontothe gold surface, thus ensuring that the aptamers were attached only tothe SAM. The R_(et) increase is caused by the electrostatic repulsionbetween the immobilized aptamer and the redox probe, causing a barrierfor the interfacial electron transfer. These results show successfulimmobilization of the SAM layer onto the gold surface and stable bondingof the aptamer to the SAM

Magnetic Bead (MB)-Based Maximum Binding Capacity

After the aptamers-coupled MBs were thoroughly washed, thrombin wasadded and the concentration change was measured using a carboxylmodified SPR sensor. The refractive index is controlled only by theconcentration change of the added thrombin. Other experimental variablessuch as protein degeneration and temperature had minor influences on SPRresults and thus were not considered to affect the results.

As shown in FIG. 3, the concentration change of thrombin wasinsignificant for the control group (less than 3%) which was notfunctionalized by the aptamer. This shows that the concentration changein the two experimental groups was mainly due to the binding between theaptamer and thrombin. For the APT1 and APT2 groups, the mixture ofaptamer functionalized MBs and thrombin solution was allowed to reactfor 18 hours and the reaction was considered to be completed based onthe MB manufacturer's specifications. Thus, the final concentrationreflected the maximum mol/mol binding capacity of aptamer to thrombin.

The results showed the binding ratio of APT1 (57.1%) has a bettercapacity than APT2 (55.2%). Both aptamers had more than 50% mol/molbinding ratio to thrombin, indicating that they are good receptorcandidates for thrombin sensing applications. It is to be understoodthat, in certain embodiments, not all the aptamers may bind to the MBsand therefore the actual binding capacity of the binding aptamers towardtarget compound/s may be slightly greater.

The Control group was comprised of MBs without aptamer functionalizationand all binding sites blocked by BSA. The aptamers-containing groupswere: APT1- and APT2-MBs functionalized by the respective aptamers withthe unoccupied binding sites blocked by BSA. The error bars representthe standard deviation of the values determined from three samples.

SPR Results

Two different aptamers were immobilized on gold surfaces and the bindingperformance of each one was compared. For reference, samples ofdifferent thrombin concentrations (5 nM, 25 nM, 50 nM, 250 nM, 1000 nM,2000 nM) were individually loaded onto a bare Au sensor, an APT1 sensorand APT2 sensor, respectively. A secondary experiment was then performedusing the same thrombin concentrations; however, with a 400 nM BSAconfounding component added to each thrombin sample for comparison. Asshown for the “Thrombin only” experiment in FIG. 4, the SPR shifts werevery low for the bare Au sensor surface even for the relatively highthrombin concentrations.

In contrast, for the aptamers-modified sensors the SPR shifts weresignificantly enhanced and the optimal detection range was 5 nM to 1000nM (linear range). The “Thrombin+400 nM BSA” data (shown in FIG. 4)shows where a large 400 nM BSA confounding concentration component wasadded to each thrombin sample concentration. As compared to the thrombinonly group, the responses are nearly identical, indicating theaptamers-modified APT1 and APT2 sensors are highly specific to onlythrombin.

This is further illustrated in FIG. 5, which shows the SPR shift for the500 nM thrombin concentration with, and without, 400 nM of BSA. AddingBSA to the sample had minimal effect on the SPR response for the aptamermodified sensors, indicating a good selectivity of the sensor towardthrombin. This is in contrast to the bare Au sensor, which experienced asignificant change between the thrombin samples with, and without, BSA.The APT1 modified sensor did have a slightly higher shift than the APT2sensor for all the thrombin concentrations. The slope of the fittingline for APT1 is also slightly larger than APT2 in the linear responserange (FIG. 6), again demonstrating a better sensitivity. These twoaptamers bind to different sites of thrombin, thus the affinity to thetarget is different in both the interfacial binding environment and insolution.

Antibody Sensing

In the MBs binding tests, the APT1 had a slightly higher bindingcapacity than APT2, which corresponds to the SPR results in terms ofsensitivity of the functionalized sensor. While not wishing to be boundby theory, it is believed that in this embodiment, this may be due tothe smaller aptamer having a greater probability to access the bindingsites of the target protein. Also, in certain embodiments, largeraptamers that have more complicated secondary structures may require anextra spatial flexibility to form bonding with target compounds.

As Example 1 herein shows, the MPA layer has excellent coverage rate ongold and is useful for antibody immunization for biosensing purposes.These results also show that the amine-modified aptamer is readilyimmobilized onto the MPA layer and the sensor performance was comparableto antibody-based sensors.

Three sensing slides were prepared for each aptamer and also the controlgroup. The sensor to sensor performance was consistent when using thefreshly prepared samples, yielding relatively small errors for eachmeasurement and averaging less than 2% standard deviation of the totalsignal (error bar showed in FIG. 5).

Adding BSA did introduce a slightly larger error and by lowering theflow rate and increasing the sample loading time, the error can bereduced although deemed not significant enough to be considered. Themajority of the error is thought to be caused by temperature variance;as such, in some embodiments, placing the sensor in a temperaturecontrolled environment can help increase the accuracy.

The sensing surface described herein had an optimal dynamic range from 5nM to 1000 nM, which is comparable to or greater than the largestreported dynamic ranges for thrombin aptamer-based sensing techniques.Since the thrombin concentration range in the human blood is reported tobe within the low nanomolar to low micromolar range, the presentlydescribed method is well suited for in vivo thrombin quantitativedetection.

Reversibility of Sensors

To test the reversibility of the sensor, fixed sample concentrationswere repeatedly loaded to the sensor 10 times. The sensor regenerationwas done by PPA. The average SPR response with error bars for standarddeviation using thrombin concentrations of 50 nM, 250 nM and 500 nM areshown in FIG. 6. All data were obtained from freshly prepared sensingslides. The SPR response generally decreased about 0.5% for each loadingfor a same sample concentration. All the sensing slides maintained morethan 95% of the original SPR shift response after the 10^(th) loading.Also, the second sample loading usually had the greatest response changeas compared to the following loadings. With a longer PPA injection time,the sensor recovery rate can be increased, depending on the experimentalrequirements. The appearance of BSA did lower the sensitivity of thesensor (e.g., in FIG. 6, the appearance of BSA did reduce the slopeslightly in the response curve), although it did not affect thereversibility of the sensor. FIG. 6 also demonstrates that sensormaintained a linear response with and without the appearance of BSA inthe 50 nM to 500 nM sample range.

Example 2 Other Embodiments of Sensors

In another embodiment, the sensor can include a mixed length spacerlayer. In one non-limiting example, the mixed length layer can be as11-mercaptoundecanoic acid (MUA) combined with MPA, which can be used incertain embodiments to increase the sensitivity and specificity.

In other embodiments, a mixed length spacer can be included to help formand maintain the specific shape of the immobilized aptamers.

In another embodiment, a hydrophilic group such as ethylene oxide can beinserted onto the 5′-end of the aptamer in order to reduce nonspecificprotein binding.

In certain embodiments of the two step immobilization method describedherein, spacing the aptamers can also done by adjusting the MPA SAMdensity, or by co-incubating ethanolamine and the aptamer at variousmolar ratios.

Detection of Blood Proteins

For the detection of different blood proteins, in order to find theaptamer that specifically and directly binds to the target protein ofinterest, a SELEX procedure can be used. Then, the developed aptamer canthen be amine-terminated and immobilized onto the gold surface using oneof the presently described methods in order to form a target specificsensor for almost any protein. As such, aptamers can be generatedthrough SELEX to target specific compounds with advantages overantibodies.

The two-step immobilization method described herein is especially usefulfor the immobilization of a SAM and amine-terminated aptamer onto a goldSPR sensing surface. The presently described SPR sensor providesadvantages, such as low sample consumption, the lack of labelingrequirement, high sensitivity, and fast response time. Additionaladvantages of the two-step immobilization method include demonstrablecost efficiency, good reversibility, uniform density, and use as arobust and specific blood protein detection platform.

Example 3 SPR Aptamer Based Glycated Albumin Protein Sensing

Glycated human serum albumin (HSA) was both detected and quantified. Theaptamer (thiolated, non-reduced) developed and used was5′-SH—(CH₂)₆-CCGAAACCAGACCACCCCACCAAGGCCACTCGGTCGAACCGCCAACACTCACCCCA-3′[SEQ ID NO: 3].

Gold slides were prepared by physical vapor deposition (PVD) forming a 1nm layer of titanium and a 50 nm layer of gold onto pre-cleanedmicroscope cover slides. The gold slides were then washed by copiousamounts of DI water and ethanol. The gold slides were dried in nitrogengas before usage.

The thiolated aptamer was diluted by 1M phosphate buffer pH 8 and mixedwith Cleland's REDUCTACRYL™ reagent in a shaker for 2 hours to reducethe double thiol bond in the aptamer sequence. Cysteine is a watersoluble thiol-containing amino acid that can directly bind to the goldsurface to form a self-assembly monolayer (SAM), which was then added tothe aptamer solution to help space out the aptamers, fill the gapsbetween aptamers, and reduce the non-specific surface absorbance. Thefinal concentration of the aptamer in this preliminary experiment wasset to be 1 μM and the aptamer:cysteine molar ratio was 1:10. The goldslides were immersed in the aptamer/cysteine mix solution at 37° C.

After the immobilization process, the gold slides were washed with 0.01M PBS buffer pH 7.4. The functionalized surface was then coupled to thecorresponding SPR sensor, and 1 μg/mL total protein HSA samples (i.e.,total=glycated+nonglycated) were prepared for the given glycated percent(%) ratios (glycated/total protein): 2, 6, 10, 14, and 18%.

SPR responses were recorded for each respective sample. The results forthe functionalized surfaces along with the bare-Au surfaces aresummarized in FIG. 10. The aptamer functionalized SPR surface respondsdirectly to changes in the glycated protein content. It is to be notedthat the total protein concentration is constant at 1 μg/mL betweensamples.

The non-functionalized surface (i.e., bare gold) exhibits a negligibleresponse, further illustrating the enhanced sensitivity in thefunctionalized surface. Although small in length (40-60 nt), in certainembodiments, aptamer sequences may differentiate targets based on sizeand charge, and affinity may be affected. While not wishing to be poundby theory, the inventors herein now believe that the 3D structure of theaptamers may also plays a role; one non-limiting examples include thecytosine-rich bulge-loop structure and the ACC(C) or (C)CCA motifs.

Aptamers for Non-Glycated and Glycated Protein Binding Sites for HbA1c,Albumin, and IgM

Aptamers were developed to attach to the self assembled monolayers(SAMs). For certain embodiments, the proteins hemoglobin, albumin, andIgM are useful since half-life of each provides information that spansshort, intermediate, and long term historical records in glycemiccontrol. A summary of the properties for some common blood proteins areprovided in Table 1 below.

TABLE 1 Related properties of blood proteins Target Half-life AverageConcentration Percent Glycation Protein (weeks) (mg/mL) (%) Hemoglobin6-8 325  6-15 IgG 3-4 12 20 Albumin 3 33 16 IgM 1 1.4 15-35 Fibrinogen0.5 2.5  6

Glycation of the respective proteins can be performed by incubation (37°C.) of the respective proteins in pH 7.4 PBS containing 1M glucose andDTPA for two days. The glycated proteins are then subjected to adialysis process and then can be further enriched by affinitychromatography. In this step, the glycated proteins can be separatedfrom the respective non-glycated forms using boronic acid immobilized onpolyacrylamide beads in the support column. Through this process, boththe nonbound and bound fractions can be collected and furtherconcentrated using filtration methods.

To achieve isolation and identification of key oligonucleotides(aptamers) specific to hemoglobin, albumin, and IgM in both the glycatedand nonglycated versions of the proteins, an improved SystematicEvolution of Ligands by Exponential (SELEX) enrichment method can beused, as further explained below, and schematically illustrated in FIG.12.

The improved SELEX method allows for the screening of ligands that areparticular to a protein of interest. The improved SELEX method can beconducted by generating a large library of randomized RNA sequences.This library usually contains 10¹⁴-10¹⁵ different RNA species that foldinto different structures depending on their particular sequence. Thislibrary is then incubated with the target protein of interest, and thoseRNAs contained in the library that bind the protein are then separatedfrom those which do not. The retained RNAs are then amplified by RT-PCRand transcribed in vitro to generate a pool of RNAs that have beenenriched for those that bind the target of interest. This selection andamplification process can be repeated between 8 to 12 rounds until theRNA ligands with the highest affinity to the target protein areisolated. These aptamers are then cloned and sequenced.

Determination of Ratio of Glycated Protein-to-Total Protein

The percent ratio of glycated protein to total protein measurement wasrelated to average blood glucose over a given time window.

Aptamers specific to the glycation sites of the target proteins can begenerated. Also, aptamers that will bind both the glycated andnon-glycated versions of the respective proteins were generated. In oneembodiment, glycated versions of hemoglobin, albumin, and IgM proteinswere used as the target in the SELEX protocol. The resulting reducedaptamer pool contains both the non-glycation site and glycation-sitespecific aptamers. At this point and in a later round(s), non-glycatedprotein (i.e., normal protein) can then be introduced, in which, presentaptamers that recognize the glycation site do not bind and can berecovered for characterization. This method provides separate aptamersthat are capable of binding both the glycated/nonglycated versions ofthe proteins, as well as those that are only specific only to theglycated versions.

Optimization of Surface Plasmon Resonance Self Assembled MonolayerAptamer-Based Functionalized Surface

The identified aptamer can then be initially characterized for generalperformance including binding properties, sensitivity, specificity, andselectivity. Presented in Table 2 below are examples of targetspecifications based on performance levels.

TABLE 2 Specification Parameter Detection Limit Hemoglobin 10⁻⁷ molAlbumin 10⁻⁶-10⁻⁵ mol IgM 10⁻⁸-10⁻⁷ mol Cross-Reactivity <6% Assay Time<15 min

In particular, one method for characterizing binding affinities is theuse of a SPR method. Based on the aptamer candidates identified, SPR isuseful to generate the respective binding response curves. For example,certain devices (such as SensiQ, iCx Nomatics) are equipped with a dualmicrofluidic channel and have controllable flow rates. The tests can beperformed using immobilization methods similar to those described forFIG. 1.

Modifications to Facilitate Immobilization

Also, in certain embodiments, the glycated and non-glycated specificaptamer candidates can be modified with a 5′-NH₂—C₆ attachment tofacilitate immobilization onto a —COOH modified gold SPR surface. SPRmeasurements are then used to characterize the respective affinityconstants for the aptamer candidates.

In addition to the affinity tests, using the SPR chip immobilizedaptamers, both the specificity and selectivity can be evaluated. In suchembodiments, the respective aptamer chips were exposed to each targetprotein in both the glycated and non-glycated forms. Cross-reactivitybetween the two forms for a given protein, as well as, for differentproteins (e.g., albumin for a HbA1c aptamer chip) was thus determined.In certain preferred embodiments, the target cross-reactivity is desiredto be below about 6%. If it is determined that this criterion is notmet, the SELEX protocol can be repeated with improved selectionconditions (e.g., increasing the frequency of elimination rounds), inorder to further improve cross-reactivity performance.

It is also understood that good target recognition can also be affectedby the aptamer linking process used for immobilization. In certainembodiments, the method can include the use of one or more alternativelinking methods of the aptamers. In certain embodiments, the linkagescan be through 3′-amino, thiol, or other potential linkages.

It is also within the contemplated scope that such linkages can bemodified by, for example, controlling certain parameters, such as thedensity and length. Thus, aptamers and linkage methods can be optimizedto provide maximum desired performance. In addition, the methoddescribed herein to create the functionalized surfaces can be optimizedto provide a desired level of uniformity in the surfaces, as well as tooptimize the aptamers sensor response.

Self-Assemble Monolayer (SAM) Linkages

In addition to the linking methods described above, another method thatcan be used include linkage through a binary self-assembled monolayer(SAM) and reductive desorption process. Since SAMs' packing density andlengths of SAMs affect the SPR signal, the density and length of thebinary SAMs can be controlled using a reductive desorption process.

In a particular embodiment, synthesized dithiobis-N-succinimidylpropionate (DTSP) can be used with (1-mercapto-11-undecyl)tri(ethyleneglycol) (PEG3) for tailoring a mixed SAM. PEG3, which is resistant toprotein adsorption, can be employed to prevent non-specific adsorptionof proteins. In addition, the carboxylic group in DTSP will form astable bonding with the aptamer.

In a particular embodiment, a thiol SAM immobilization method usingdithiobis-N-succinimidyl propionate (DTSP) was used in a phosphatebuffer solution. DTSP is useful for SAMs due, at least in part to itsdistinctive surface properties, such as hydrophilicity, wettability,chemical reactivity, and an affinity towards proteins such as hemoglobinand cytochrome c.

For the binary-SAM immobilization, 3-mercaptopropionic acid (MPA) and(1-mercapto-11-undecyl)tri(ethylene glycol) (PEG3) can be used. Incertain embodiments, MPA is chosen because it has a lower redoxpotential than PEG3, which means MPA can be easily eliminated byreductive desorption leaving PEG3 intact. DTSP is able to form acovalent bond with the amino groups of the aptamer while PEG3 does not,so that the aptamer will attach to DTSP only.

Two-component thiol solutions can be prepared by mixing 1 mM ethanolsolutions of MPA and PEG3 at various ratios, while keeping the totalconcentration of the binary SAMs at 1 mM. The binary SAM of MPA andPEG3, whose ratios are 20:80, 50:50 and 80:20, can then be formed on agold electrode by soaking the electrodes into the mixed thiol solutionfor 1 hr.

Referring now to the schematic illustration in FIGS. 11a-11e , a binarySAM formation and reductive desorption procedure is shown. First, thebinary components of 3-mercaptopropionic acid (MPA) and PEG3 areadsorbed on the gold surface in an ethanol solution (FIG. 11a ). Thereductive desorption of MPA from the gold electrode is performed in 0.5M KOH solution. The adsorbed MPA in a phase-separated binary SAMs of MPAand PEG3 is selectively reduced by applying the potential of −1.2 V for30 min (FIG. 11b ).

After reductive desorption of MPA, the sample with the PEG3 layer isimmersed in the 1 mM DTSP solution to form DTSP layers (FIG. 11c ). FIG.11d shows aptamer immobilization, and FIG. 11e shows removing aptamerfrom PEG3.

The aptamer covalently couples to the SAM of DTSP exposing —COOH endgroups. For covalent bond formation, aptamer (50 μg/ml) in PBS isinjected together with freshly prepared NHS and EDC. Aptamers havingamino groups at the N-terminals and can be immobilized on the DTSP SAMthrough CO—NH amide bond formation. The ratio of DTSP and PEG3 will bevaried to control the packing of the SAMs and as result, the binding ofthe protein that gives the optimum SPR signal can then be obtained.

Measurement of Surface Coverage

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS)can be used to measure the surface coverage of immobilized SAMs andredox responses of the samples. The surface composition can be estimatedfrom the peak areas of a cyclic voltammogram for the adsorbed thiols.Responses of the binary SAMs deposited on the modified electrode can becompared with those of the unmodified electrode.

Cyclic voltammogram of the reductive desorption can be recorded in 0.5mol dna⁻³ phosphate buffer solution using a Ag—AgCl-saturated KClelectrode as the reference electrode and a platinum wire as the counterelectrode. The CV curves of SAMs+Aptamer coated gold electrode(Au+SAM+aptamer) and reductive eliminated SAMs and aptamer on the goldelectrode (Au+RD SAM+aptamer) can thus be compared. The CV curves can berecorded at the scan rate of 100 mV/s for the reductive elimination. Ineach voltammogram, a down peak of reductive desorption of SAM isexpected to appear around 50 mV.

Both the length and density of the SAM can be controlled to obtain theoptimal SPR response. When the linker length is long, more aptamers canbe immobilized, but the SPR dip may get broader as the aptamers arefarther away from the surface. Likewise, when the linker density ishigh, more aptamers can be attached to the SAM, but then the SPR dip mayget narrower and more difficult to detect. These aptamer modifiedsurfaces can be characterized by the methods used with the5′-NH₂—C₆/—COOH method.

Calibration and Validation of the Developed Functionalized SPR SensingSurfaces

The SPR sensing platforms for HbA1c, albumin, and IgMglycated/non-glycated protein detection can initially be calibrated intests using a saline buffer with known target proteins ratios.Respective sample solutions can be prepared for fixed levels of totalprotein at reasonable ratio levels compared to those seen in blood (seeTable 1).

For each sample, the ratio of glycated protein to the total amount ofprotein can be varied over a desired range (e.g., for HbA1c % levelsbetween 6 to 15% correspond to average glycemic levels of 60 to 360mg/dL, respectively). In such embodiment, a range from 1 to 25% v/vwould be appropriate. The SPR response in the respective samples canthen be evaluated and a calibration model can be determined in relationto % glycation and the standard error of calibration can be calculated.To further assess the accuracy of the developed SPR assays, independentsamples (i.e., those not used in calibration) can be used to assessassay performance based on the respective calibration model(s). Bothrelative and absolute errors can be determined and compared with theranges that would be required for useful diagnostic purposes.

Testing of Serum Blood

To assess performance in actual blood serum, blood serum from anon-diabetic source can be utilized. The serum samples can be analyzedto determine the respective fractions of glycated versus total protein(for both protein targets) through standard clinical testing.

Using these values as references, individual samples can be doped withspecific amounts of the respective glycated protein(s). Testingevaluation similar to that utilized with the saline tests can berepeated. It is understood that, due to high concentrations of certaintarget proteins in serum (e.g., hemoglobin as shown in Table 1), it maybe desired to dilute the samples prior to running the tests. Inaddition, other potential confounding effects such as introducingvariations in sample composition, outside that of glycated protein, canbe tested as issues may arise due to the complex chemical composition ofserum.

Example 4 Improved SELEX Method for Aptamer Identification Targeted toGlycated and/or Non-Glycated Protein Sites

The SELEX protocol was improved in order to allow for the identificationof aptamers with an affinity to glycated protein sites. This improvedSELEX protocol allowed for the determination of the percent ratio ofglycated protein to total protein.

Aptamers specific to the glycation sites of the target protein(s), inaddition to those that will bind to both the glycated and non-glycatedversions of the respective proteins, were generated. In order togenerate such aptamers for a respective protein (e.g., hemoglobin,albumin, IgM, etc. . . . ), in a first round of amplification, the SELEXprotocol was applied to a glycated version of the respective proteins.This first round of the SELEX protocol resulted in a reduced aptamerpool that contained both “non-glycation-site-specific” aptamers and“glycation-site specific” aptamers.

A non-glycated protein (i.e., normal protein) is introduced into thepool obtained in the first round SELEX amplification process. In atleast a second round of amplification, the aptamers in the pool thatbind to such non-glycated protein are not eluted in this specific SELEXround, and are, therefore, are removed from the pool. This improvedSELEX protocol improves the chance that aptamers specific to theglycated sites will remain in the ongoing pool. Such remaining aptamerscan then be recovered for characterization in subsequent SELEX rounds aspart of a standard SELEX process. It is to be understood that, in otherembodiments, the uses of “glycated” protein and “non-glycated” proteincan be reversed; e.g., where a “glycated” protein is introduced onto thepool obtained in the first round SELEX amplification process.

Determination of High Affinity Glycated and/or Non-Glycated ProteinAptamers

A protein molecule (e.g. albumin) has multiple sites available forglycation. The glycation level usually refers to the percentage of agiven protein concentration that has been glycated with respect to thetotal protein level, whereas, the glycation rate refers to how manysites within a single protein molecule has bound glucose or glucosederivatives. The 3D conformation and the charge distribution aresignificantly different between a highly glycated and non-glycatedprotein molecule, but very similar between a lightly glycated proteinmolecule (i.e., single glycation point) and non-glycated proteinmolecule. Therefore, the development of a high affinity single-sitespecific glycated protein binding aptamer that has a low affinity to thenon-glycated form is very challenging.

One example of the improved SELEX in vitro selection protocol is shownin FIG. 12, where a large random DNA pool is initially mixed with aglycated protein target immobilized onto magnetic beads (MBs); that is,a primary or “glycated-protein-target-MB” complex.

Aptamers with high affinities to the glycated protein target will bindand form an “aptamer-glycated-protein-target-MB complex.”

The “aptamer-glycated-protein-target-MB” complex is separated out fromthe initial DNA pool.

In a subsequent step, the bound aptamers are eluted from the“glycated-protein-target-MB” complex (i.e., the single or lightlyglycated form of the protein).

At this point, a control protein (i.e., a non-glycated form of theprotein), which is coupled to a second set of MBs (a secondary or“non-glycated-protein-target-MB” complex) is added to this firstelution.

The “non-glycated-protein-target-MB” complex is used to remove“selective” aptamers in the first elution that also have an affinity tothe non-glycated protein form.

In a subsequent step, the “selective” aptamers are eluted from the“non-glycated-protein-target-MB” complex.

Upon the removal of the “non-glycated-protein-target-MB” complexes via,the remaining “selective” aptamers are those aptamers that have a highaffinity to the single or targeted glycation sites.

At this point, a standard SELEX method can be used to amplify theseremaining “selective” aptamers that have a very high affinity to thedesired glycated protein site.

Specifically, this improved SELEX method allows for the development ofhigh affinity single glycation site aptamers that have a low affinity tothe non-glycated form of the protein. This improved SELEX method is alsouseful to generate aptamers that have an ability to distinguishanalytes/molecules that have very similar chemical structures.

Examples of Glycated and Non-Glycated Aptamers

Examples of useful aptamers are shown below, where XXX and YYY refer toany one or more of additional binding groups such as biotin, thiol,amine, etc. that may be used to facilitate development of a givenself-assembly-monolayer (SAM).

Glycated Hemoglobin Aptamers

[SEQ ID NO: 4] 5′-XXX-ATCCTTCATCCCATGGTTGCATATTGATTGCCGGTTCCTTAAAT-YYY-3′; and [SEQ ID NO: 5]5′-XXX-AGGGAAAGGTGTGGGTTAGGAGCTTGAAATCGAA AAGAGGGGCG-YYY-3′.

Non-Glycated Hemoglobin Aptamers

[SEQ ID NO: 6] 5′-XXX-TTAGCGAGCTGCACACACAATGGACTCGTCATACCGTGCTGTTT-YYY-3′; and [SEQ ID NO: 7]5′-XXX-ATCTGCAGAATTCGCCCTTGCTGGTGCAGTACAC ACCCGGCGGG-YYY-3′.

Glycated: Human Serum Albumin (HSA) Aptamers

[SEQ ID NO: 8] 5′-XXX-CTCACTCCATACTCACTTGCTGATTCGCCAACAACACACCCTTAAACAGTC-YYY-3′; and [SEQ ID NO: 9]5′-XXX-CCGAAACCAGACCACCCCACCAAGGCCACTCGGT CGAACCGCCAACACTCAC-YYY-3′.

Nonglycated: Human Serum Albumin (HSA) Aptamers:

[SEQ ID NO: 10] 5′-XXX-CTCTCCGGCCGCTGACCCAGTTTGGAGGGGGGAGGAGGCCGGGC-YYY-3′; [SEQ ID NO: 11]5′-XXX-ACGGGCACTGGTTCCATCCGCATGAGATTGATGT GTCAACTTAT-YYY-3′;[SEQ ID NO: 12] 5′-XXX-CAATACCGATTGTTCTAAGGGAAAACGTGTAACTTTGGATCCTT-YYY-3′; [SEQ ID NO: 13]5′-XXX-TAGCGACACACGTGGCCGCTGGTTGCCGGGCGCC ACGGATCCTT-YYY-3′;[SEQ ID NO: 14] 5′-XXX-CCAGCTCGTAGTGGCGTCTTTTTTTCATTTGGTACTTATCGCAA-YYY-3′; and [SEQ ID NO: 15]5′-XXX-AAATTTCATGTTCCCACACGTTCCATGCGCCCTC CTTCGAGTGC-YYY-3′.

Example 5 Surface Functionalization Method Using SAMs for OptimizingSensitivity and Selectivity Based on Target Characteristics

The sensitivity and selectivity of the binary SAM formation for aptamermobilization may be further enhanced. For example, to control thelinking spacing and the distance between the aptamer and SPR surface,two different types of self-assembling thiol molecules are deposited onthe surface. A 1 mM ethanol solution of 11-mercaptoundecanoic acid(SH—(CH₂)₅—COOH, MUA) and mercaptopropanol (SH—(CH₂)₂—OH, MPL) areprepared separately. Each solution is mixed at a 1:1 volume ratio whilekeeping the total concentration of the two components at 1 mM. A binarySAM of MUA and MPL is formed on a gold surface by soaking the goldsurface in the mixed thiol solution for 1 hr. Then, the gold surface issubsequently rinsed with ethanol and DI water.

MPL density can be controlled for optimum signal transfer by applying anelectric potential to the gold surface in 0.5 M KOH solution (pH 13).The applied potential of −0.5˜-1.0 V for 30 min detaches portion of MPL,resulting in a less dense MPL layer that enhances signal transfer. Then,the surface is immediately washed by DI water.

After the surface is dried, then it is treated with a solution ofN-hydroxysuccinimide (NHS) andN-(3-dimethylamnopropyl)-N-ethylcarbodiimide hydrochloride (EDC) (NHS0.2M, EDC 0.05M) for 30 min to activate the carboxyl group of MUA. Thesurface is then washed with DI water and then immersed in the 5 μMaptamer solution. Aptamers are covalently attached to the activated MUA.Finally, the surface is rinsed with the PBS buffer.

This surface functionalization method is applicable not only for SPR,but also to optimize the sensitivity and selectivity of other sensingmodalities such as Raman and fluorescence spectroscopy. The method canbe used to improve the performance of existing monitoring technologies.

Example 6 Methods for Reducing Effects of Confounding Substances Presentin Samples

As part of the functionalization process, the MPL layer is hydrophilicin nature. This property can prevent the non-specific adsorption ofproteins to the surface. In another embodiment, the aptamer recognitionelement can be extended beyond the normal SPR sensing range (while stillmaintaining a desired sensitivity) through an extended linking approach.In this embodiment, multiple linkages can be obtained throughterminations, such as for thiols. Between the terminations, goldnanoparticle interfaces can be made by exposing the surface to a goldnanoparticle solution. This nanoparticle coupling can allow the aptamerbinding response to be detected by the SPR sensor at separationdistances beyond the normal SPR detection limit.

It is to be noted that, as in the non-aptamer locations, densely packedlinkages of lengths outside the SPR range can be made that are void ofmetal particle coupling. Therefore, if non-specific protein adsorptionor other confounding components are encountered in these locations, acorresponding SPR response will not occur, thereby improving theselectivity performance for the sensor.

In another embodiment, a secondary physical vapor deposition (PVD) canbe formed over subsequent MPA layers, followed by thermal treatment toobtain a similar structure to extend the aptamers away from the SPRfoundation surface, while maintaining sensitivity through the metalcoupling linkages.

Example 7 Biomarker Detection

The method and platform described herein are also useful in the field ofbio marker detection for disease diagnosis and assessment.

For example, for the proteins described herein (e.g., glycatedproteins), the accurate detection can facilitate the treatment ofdiabetes and help minimize the numerous associated healthcareconditions, such as increased risk of cardiovascular disease, blindness,kidney failure, and many others.

The method and platform herein can be miniaturized so as to be easilyintegrated into a handheld device, thus allowing the method and/orplatform to be used directly in physician offices, in the home, or inthe field.

The measurements of glycated proteins (which are a measure of glycemiccompliance), instead of being only available during physicianexaminations through untimely offsite analysis, are thus readilyavailable to the patient or healthcare giver in a more readilyassessable manner. These more widely accessible measurements would, inturn, provide complimentary information to that of self-monitoring bloodglucose measurements to further help diabetics better manage theircondition and mitigate potential long term health complications.

Furthermore, if such information is available on a more frequent basiswith expanded historic time windows, this could significantly impact theunderstanding of glucose regulation within and outside the diabeticcommunity, which could lead to a better understanding of glycemiccontrol through the development, education, and training of new and/oroptimized therapeutic approaches to diabetes.

Example 8 Kits

The sensor described herein can be provided in the form of kits ofparts. Such kits include but are not limited to diagnostic kits,biomarker discovery kits, environmental testing kits, biohazard orbioweapons detection kits, and kits for detecting targets in medical oranalytical chemistry applications. By way of non-limiting example, theamine-terminated aptamers can be included as a molecule alone or alreadyattached to a substrate. Additional components can also be included andcomprise microfluidic chip, reference standards, and additionalcomponents identifiable by a skilled person upon reading of the presentdisclosure. Also, the components of the kit can be provided, withsuitable instructions and other necessary reagents, in order to performthe methods here disclosed. In some embodiments, the kit can contain thecompositions in separate containers. Instructions, for example writtenor audio instructions, on paper or electronic support such as tapes orCD-ROMs, for carrying out the assay, can also be included in the kit.The kit can also contain, depending on the particular method used, otherpackaged reagents and materials (such as wash buffers and the like).

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

Citation of the any of the documents recited herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

What is claimed is:
 1. A method for optimizing sensitivity and/orselectivity of a sensor for one or more analytes, comprising: linkingone or more types of aptamers to a substrate with a self-assembledmonolayer (SAM) linkage, the SAM linkage having a desired linkingspacing and/or length to form a functionalized surface on the substrate,the desired linkage spacing and/or length being chosen in order tooptimize at least one of surface plasmon resonance (SPR), Ramanspectroscopy, electrochemical spectroscopy, or fluorescence spectroscopysensitivity and selectivity based on analyte and/or aptamercharacteristics.
 2. The method of claim 1, wherein at least one packingdensity and/or length of the SAM linkage affects a surface plasmonresonance (SPR) signal.
 3. The method of claim 1, wherein linkage isthrough a binary SAM and reductive desorption process.
 4. The method ofclaim 3, wherein the desorption process comprises: exposing thefunctionalized surface of the substrate to a material resistant toprotein adsorption to inhibit non-specific adsorption of protein on thefunctionalized surface.
 5. The method of claim 1, wherein the SAMlinkage is formed using dithiobis-N-succinimidyl propionate (DTSP) and(1-mercapto-11-undecyl) tri(ethylene glycol) (PEG3), wherein PEG3prevents non-specific adsorption of proteins, and wherein a carboxylicmoiety on DTSP forms a stable bonding with the aptamer.
 6. A kit for thedetection of one or more analytes, comprising: a sensor comprising oneor more aptamers linked to a substrate with a self-assembled monolayer(SAM) linkage; and at least one container including the sensor, where asample may be added to the container.
 7. The kit of claim 6, furthercomprising one or more solid supports, one or more separating agents forseparating the sensor from an elute, and one or more reagents forseparating an aptamer from the sensor.
 8. A method of identifying asingle target-site binding aptamer from a pool of nucleic acids havingsingle-target-site-binding-aptamers andnon-target-protein-binding-aptamers therein, comprising: adding to apool of nucleic acids a single-site-target-protein-complex, wherein boththe single-target-site-binding-aptamers and thenon-target-protein-binding-aptamers present in the pool bind to thesingle-site-target-protein-complex and form asingle-target-site-binding-aptamer+non-target-protein-binding-aptamer+single-site-target-protein-complex;separating thesingle-target-site-binding-aptamer+non-target-protein-binding-aptamer+single-site-target-protein-complexfrom the pool; eluting the single-target-site-binding-aptamers and thenon-target-protein-binding-aptamers from thesingle-site-target-protein-complex; adding to the elute of the previousstep a non-target-protein-complex, wherein thenon-target-protein-binding-aptamers present in the elute bind to thenon-target-protein-complex and form anon-target-protein-binding-aptamer+non-target-protein-complex;separating thenon-target-protein-binding-aptamer+non-target-protein-complex from theelute of the previous step, leaving thesingle-target-site-binding-aptamer in the elute; and separating thesingle-target-site-binding-aptamers from the elution; optionally,further amplifying the single-target-site-binding-aptamers.
 9. Themethod of claim 8, wherein single-target-site-binding-aptamers are usedto select for one of: hemoglobin, immunoglobulin G (IgG), immunoglobulinM (IgM), and albumin.
 10. The method of claim 8, whereinsingle-target-site-binding-aptamers are selected from: SEQ ID NOs: 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and
 15. 11. The method of claim 8,wherein the single-site-target-protein is immobilized on a solidsupport.
 12. The kit of claim 6, wherein the aptamer binds to glycatedhemoglobin, wherein the aptamer comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 4 and
 5. 13. The kitof claim 12, wherein the aptamer comprises at least one chemicalmodification selected from the group consisting of: a chemicalsubstitution at a sugar position, a chemical substitution at aninternucleotide linkage, and a chemical substitution at a base position.14. The kit of claim 6, wherein the aptamer has at least 70% identity tothe entire sequence of any one of SEQ ID NOs: 4 and 5, and binds tohuman glycated hemoglobin.
 15. The kit of claim 6, wherein the aptamerbinds to non-glycated hemoglobin, wherein the aptamer comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:6 and
 7. 16. The kit of claim 6, wherein the aptamer has at least 70%identity to the entire sequence of any one of SEQ ID NOs: 6 and 7, andbinds to human non-glycated hemoglobin.
 17. The kit of claim 6, whereinthe aptamer binds to glycated serum albumin, wherein the aptamercomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 3, 8, and
 9. 18. The kit of claim 6, wherein the aptamer hasat least 70% identity to the entire sequence of any one of SEQ ID NOS:3, 8, and 9, and binds to human glycated serum albumin.
 19. The kit ofclaim 6, wherein the aptamer binds to non-glycated serum albumin,wherein the aptamer comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 11, 12, 13, 14, and
 15. 20. The kitof claim 6, wherein the aptamer has at least 70% identity to the entiresequence of any one of SEQ ID NOS: 10, 11, 12, 13, 14, and 15, and bindsto human non-glycated serum albumin.